Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. However, molecular genetics underlying HCC development remain to be poorly understood. c-Myc is considered to be a key driver oncogene for HCC. Studies have shown that c-Myc driven tumor cells depend on glutamine for survival. Studies from our laboratory demonstrated the increased glutamine catabolism in c- Myc driven liver tumors. This phenotype is associated with decreased expression of Glutamine Synthetase (Glul) and switch from GLS2 to GLS1 glutaminase, resulting in increased glutaminase activity. We also showed that depriving glutamine or silencing GLS1 induces cell death in cells derived from c-Myc-driven HCCs. However, whether glutamine catabolism or GLS1 expression is required for c-Myc driven tumorigenesis; and how glutamine catabolism is integrated into the aberrant tumor metabolic system have never been studied in vivo. In this application, we hypothesize that GLS1 is required for both initiation and maintenance of c-Myc driven liver tumors in mice. Using an innovative approach by combining hydrodynamic transfection with miR-30 based constitutive and inducible shRNA silencing in vivo, we will determine whether silencing GLS1 expression inhibits c-Myc driven HCC formation in mice (Aim 1); and whether silencing GLS1 after c-Myc tumor formation induces tumor regression (Aim 2). We will further investigate the molecular mechanisms underlying the phenotypes we observe using expression analysis, metabolic imaging and stable isotope flux assays. Altogether, our proposed study will be the first to characterize the functional significanc of GLS1 in the context of c-Myc induced tumor development using in vivo modeling. The study will provide novel mechanistic insight into the metabolic requirements in c-Myc driven hepatic carcinogenesis. It will also provide strong evidence for the development of GLS inhibitors for HCC prevention and treatment. As c-Myc is considered to be the driver oncogene in many tumors, and c-Myc induces similar metabolic changes in multiple cell types, our study is likely to have broad impact in cancer metabolism beyond HCC.